Worm Breeder's Gazette 11(5): 39
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Mutations in the unc-44 gene affect the direction of axonal outgrowth and result in fasciculation of nerves with inappropriate partners [(Hedgecock et al., Dev. Biol., 111, 158-179 (1985)]. We have cloned the unc-44 gene by transposon tagging and have partially characterized the nature of six transposon insertion mutations. Results of Southern Blot Analysis of unc-44 Mutations. The putative unc-44 insertion mutations appear to result in changes in two different regions (Sites A and B) separated by approximately 10 kb. The cDNA clones DD#PA049 and DD#PA066 are found at Site A and DD#PA013 at Site B. The unc-44(rh1013) allele is an insertion at Site A. The unc-44( rh1013) mutation is due to a 1.6 kb insertion. The presence of a DNA insertion, rather than an alteration of a restriction site, is indicated by restriction fragment size differences with either EcoRI or BamHI digests probed with cosmid clone B0350 or cDNA clone A066. The presence of a SalI site in the inserted DNA suggests that it is a Tc1 element. The rh1013 insertion is included in the region covered by the cDNA clone, A066. Southern blot analysis of two independent revertants reveals a wild type pattern with B0350 and A066 probes. The unc-44(q331) allele is an insertion at Site A. The unc-44(q331) mutation is due to a 1.6 kb insertion at a site distinct from rh1013. Again, digestion with either EcoRI or BamHI reveals a 1.6 kb insertion containing a SalI site and is consistent with a Tc1 insertion. The region of the q331 insertion is not included in the A066 cDNA clone. A single revertant (q331, q332) yields a wild type pattern. The unc-44(rh1042) mutation is associated with an insertion at Site B. The rh1042 allele is associated with a Tc1 insertion (determined by DNA sequencing) in an open reading frame. The open reading frame is included in the cDNA clone A013. The Tc1 element is not excised in the single revertant analyzed, but changes occur in a nearby restriction fragment. The unc-44(st200) and mn259 alleles cause changes at Site B. The st200 and mn259 alleles result in changes in two BamHI fragments at Site B. The size changes are consistent with one insertion of 3.2 kb or two insertions of 1.6 kb each, but the exact nature of the mutations and reversions remains to be determined. The unc-44(mn339) allele results from a 2.1 kb insertion at Site B. The mn339 allele results in the loss of a 2.1 kb BamHI fragment and the acquisition of a 4.2 kb fragment. The single revertant analyzed results from a 4.3 kb deletion (the 4.2 and 0.75 kb BamHI fragments in the mutant are replaced by a 0.8 kb fragment in the revertant). Sequence analysis of unc-44 cDNAs. DNA sequence analysis A049 and A066 clones reveals nearly identical sequences in the central region with divergence at the 5' and 3' ends. A013 does not appear to be related in sequence to A049 or A066. [See Figure 1]